The budding yeast RasGEF Cdc25 reveals an unexpected nuclear localization.
The mechanisms regulating the activity of Saccharomyces cerevisiae Ras-GEF Cdc25 are still largely unknown. While the catalytical function of the C-terminal domain has been thoroughly studied, only recently a role of negative control on the protein activity has been suggested for the dispensable N-terminal domain. In order to investigate Cdc25 localization and the role of its different domains, several fusion proteins were constructed using the full length Cdc25 or different fragments of the protein with the green fluorescent protein. Unexpectedly, even if only slightly overexpressed, the full protein was not located in the cell plasma membrane, but accumulates inside the cell and also into the nucleus. Moreover, the endogenous Cdc25, tagged with HA, was also found in purified nuclear extracts. The fusions spanning aa 353-875, aa 876-1100 or aa 353-1100 localize heavily in the nucleus, concentrating in the nuclear peripheral area, in a region distinct from the nucleolus. This could be related to the presence of two predicted nuclear localization signals (NLS) in positions 547 and 806, but also to the contribution of another region, spanning residues 876-1100. This localization is likely to be physiological, since the fusion proteins can be efficiently exported and then imported back into the nucleus.
Tisi
Renata
R
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
Belotti
Fiorella
F
Paiardi
Chiara
C
Brunetti
Francesco
F
Martegani
Enzo
E
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D020666:ras-GRF1; D049452:Green Fluorescent Proteins